A major function of adjuvants is to drive maturation of dendritic cells (DC) into competent antigen-presenting cells (APC), a requirement for the generation of acquired immunity against pathogens. Double-stranded RNA (dsRNA), a molecular signature of viral infection, activates DC by binding to Toll-Like Receptor 3 (TLR3) and initiating a variety of inflammatory responses. TLR3 triggers a unique activation pathway. We therefore hypothesized that antigen-bearing DC activated by dsRNA would generate an acquired response different from DC activated with other PAMPs, and more appropriate for counteracting viral pathogens. Based upon our previous observations that the capacity of different length dsRNA oligonucleotides (ONs) to activate TLR3 depended upon levels of TLR3 expression in HEK293 transfectants, we asked how DCs would respond to different length dsRNA ONs. We therefore investigated whether homogeneous dsRNA ONs ranging in length from 25 to 540 bp, could induce DC maturation and serve as adjuvants in mice. Using a novel anti-TLR3 monoclonal antibody, we showed that two cross-presenting DC subsets, CD8alpha+ resident and CD103+ migratory DC, express much higher amounts of TLR3 protein than other cells and that TLR3 can serve as a marker for cross-presenting DC. dsRNA ONs induced CD86, IL12p40, IFN-beta, TNF-alpha, and IL-6 expression and matured DC into APC that cross-presented exogenous antigens to CD8+ T cells. By comparing DCs from wild type and TLR3-deficient mice we found that TLR3 was essential for activation of DC by dsRNA ONs and the potency of dsRNA ONs increased with length and varied between DC subsets. In vivo, dsRNA ONs served as adjuvants for the generation of antigen-specific CTL responses and induced protection against lethal challenge with influenza virus when given with influenza nucleoprotein as immunogen. Thus, dsRNA ONs of defined lengths may serve as targeted, specific adjuvants capable of inducing immune responses tailored for viral pathogens. In ongoing studies we have successfully coupled dsRNA of different lengths to protein antigens and are in the process of investigating the capacity of these complexes to trigger immune responses.